Introduction
Diarrheal diseases account for majority of deaths globally making it the second leading cause of death in childhood.1 In India diarrhea accounts to 14% deaths in children less than 5 years of age.2
Diarrhea could be due to various causes like viruses, bacteria, protozoa and helminths. In developing countriesb50-60% cases are bacterial in origin were Escherichia coli amounts to 25%, Campylobacter jejuni 10-15%, Salmonella species 5%, Shigella species 5%, followed by 35% viral etiology, were rotavirus accounts for 15-25% and in many the cause is unidentified or mixed.3
In India, acute diarrheal disease accounts to 8% deaths in children under 5 years age. During 2013, about 10.7 million cases with 1535 deaths have been reported in India.4 Although acute diarrheal disease is usually self limiting, infectious source needs proper work up and treatment.
Rotavirus infection is most common amongst the viral etiology leading to 20-30% hospitalized cases in India under 5 years age and estimated to cause approximately 5,27,000 deaths each year.5
Hence the present study was taken up for rapid diagnosis of rotaviral infections, to prevent the irrational use of antibiotic therapy and prolonged hospitalization.
Materials and Methods
The present study was a cross sectional, prospective study conducted at the department of Microbiology, from December 2015 to November 2016.
Source of data
Stool samples from patients admitted in the paediatric ward of were received at Department of Microbiology.
Methodology
A detailed clinical history was taken, thorough clinical examination was done and duly recorded in the case record forms. An evaluation of degree of hydration was done as per WHO criteria.
Subsequently stool sample was sent to department of microbiology for ELISA for Rotavirus detection.
Principle
Solid phase sandwich type EIA. Plastic microtitre wells are coated with a monoclonal antibody directed against the product of the sixth viral gene (VP6), which is the group specific antigen for all known human rotaviruses.
Procedure
Specimen preparation- Add 1 ml of sample diluent to a marked tube, using a transfer pipette.
Snap off sufficient number of wells for samples and the controls. Insert into the microtitre well holder.
Add 2 drops(100 µl) each of diluted fecal sample, positive and negative control to the bottom of separate wells.
Add 2 drops(100µl) of enzyme conjugate to each well. Mix by gently swirling on the table top.
Incubate at room temperature for 60 minutes.
Pour the liquid out of the wells into a discard jar. Tap the microtitre plate holder upside down vigorously against the absorbent paper to ensure complete removal of liquid from the wells.
Fill all the wells to overflow with deionised water and pour the liquid out as in step 5
Repeat the washing procedure four times (a total of 5 washes)
Add 2 drops (100 µl) of substrate solution to each well
Add 2 drops(100µl) of chromogen solution to each well
Incubate 10 minutes at room temperature
Spectrophotometric procedure :
This can be determined by adding 2 drops of stop solution (sulphuric acid) to each well.
Interpretation of result
Positive results by visual determination: Any sample with blue colour more than intense than that of negative control is considered positive.
Positive results by spectrophotometric determination: Specimens with absorbance unit A(450) greater than 0.150 are considered positive.
Statistical analysis of data was done using IBM SPSS statistical software version 22.
Results
A total of 100 cases of acute diarrhoea in children less than 5 years, who were admitted in paediatric department, KIMS Hospital, Hubballi, were studied during the December 2015 to December 2016.
Table 1
Showing age and gender distribution of diarrhoeal cases and its comparison to rotavirus positive cases
|
Age in months |
Male |
Female |
Rotavirus positive |
|
Less than 6 |
06 |
04 |
02(6.89%) |
|
6-12 |
24 |
24 |
16(55.17%) |
|
1-2 years |
17 |
09 |
09(31%) |
|
>2-5years |
07 |
09 |
02(6.89%) |
|
Total |
54 |
46 |
29 |
Observation
Maximum rotavirus positive cases were seen in the age group of 6-12 months (55.17%), followed by 1-2 years (31%) and least were seen among less than 6 months and 2-5 years. Also rotavirus positive cases in males and females shows 62.06% and 37.93% respectively.
Table 2
Showing the urban - rural distribution of rotavirus cases
|
Place |
No. of. cases of Rotavirus |
Non Rotavirus |
|
Urban |
10(34.48%) |
20 |
|
Rural |
19 (65.5%) |
51 |
The chi-square statistic is 0.3909. The p-value is .531852. This result is not significant at p<0.05.
Observation
The Table 2 shows that rural rotavirus positive cases are 65.5% when compared to urban population which shows 34.48%.
Observation
Above Table 2 that majority of the rotavirus positive cases were seen in the winter season which accounted to 85.37%, 10% in rainy season and 3.44% in summer season.
Observation
Above Table 2 that maximum number of cases (75%) shows rotavirus positive when the children lack exclusive breast feeding whereas 24.13% in children who were exclusively breast fed.
Observation
The Table 3 shows that samples having history of passing >10 stools per day had 55.17% of rotavirus positivity, followed by 27.58% when stools are <10 in number and 17.24% when it is less than 5 stools/day
Observation
Shows that almost 51.72% cases of rotavirus had complaints of diarrhoea, fever and vomiting, followed by 20.6% with diarrhoea and fever, 17% with only diarrhoea and 10.34% with diarrhoea and vomiting.
Observation
Maximum number of cases of rotavirus positive samples were showing some dehydration(58.62%), followed by equal distribution of cases in no dehydration and severe dehydration which had 17, 6 and 6 respectively. Among severe dehydration Rotavirus cases were 66.66%.
Observation
Above Table 5 that 70% cases were presenting with yellowish coloured stools, followed by 22% showing greenish stools and 8% with solid stools1showing macroscopic picture of greenish frothy stools with mucous flakes of rotavirus positive specimen:
Figure 1
Showing macroscopic picture of greenish frothy stools with mucous flakes of rotavirus positive specimen
Discussion
This study was conducted in the department of microbiology from December 2015- December 2016.
The total number of acute diarrhoeal cases admitted to paediatric ward during this period were 186 in accounting to prevalence rate of 5.86% in our set up.
Among them, 100 cases were included in the present study for acute diarrhoea which were within our inclusion criteria.
In the present study maximum rotavirus positive cases were seen in the age group of 6-12 months (55.17%), followed by 1-2 years(31%) and least were seen among less than 6 months and 2-5 years. Also rotavirus positive cases in males and females shows 62.06% and 37.93% respectively. Also in a study conducted by Satarupa Mullick et al 6 in 2014 most cases of ADD belonged to 6months to 2 years, followed by 0-6months of age. In another study done by Shaveta Dhiman et al 7 in 2015 at Amritsar maximum cases of ADD (69%) belonged to 6 months - 2 years of age. These findings were similar to the present study. Predominant number of cases in the age group of 6-12 months may be due to the commencement of weaning period, which leads to exposure to the water and other contaminants and also during this period infant starts toddling and acquire the habit of mouthing. 8
Table 6
Showing commonly affected age group due to rotavirus infection
|
S.No |
Author |
Year |
Place |
Total no. of study cases |
Commonly involved age group |
|
1 |
Shaveta Dhiman et al 7 |
2015 |
Amritsar |
100 |
6months-2 years |
|
2 |
Anup kumar Shetty et al 9 |
2014 |
Karnataka |
35 |
7months- 2 years |
|
3 |
Ma Mathew et al 10 |
2013 |
Kerala |
1827 |
6 months to 2 years |
|
4 |
Muhammed Irfan Habib et al11 |
2007 |
Karachi |
300 |
6months-2 years |
|
5 |
Pierre Van Damme et al12 |
2005 |
Europe |
2846 |
6months-2 years |
|
6 |
Present study |
2016 |
Hubballi |
100 |
6months-2 years |
In the present study out of 100 children presenting with acute diarrhoea 54% were males and females accounted for 46%. In a study at Tumkur, Karnataka, from Nov 2007 –Aug 2009, the prevalence of diarrhea in children accounted for 10.12%. Out of 150 cases enrolled, maximum cases of diarrhea occurred in the first 24 months of life 107 (71.3%), followed by 24-60 months (15.3%) and 6-12 years (13.3%).13 In a study conducted by Manohar Badur et al in 2015 in Tirupati, predominant number of males were affected which is around 57.2% and females were 42.8%.14 Thus the results are almost similar to the present study. The rotavirus cases in present study accounted for a total of 29%, with a predominant male pattern with 62.06% and females showing 37.93%, showing ratio of 1.5:1. In a study conducted at Lucknow during August 2010-July 2012, rotavirus was the most frequent cause of viral gastroenteritis in both hospitalized and non-hospitalized children accounting for 19.5% and 9.1% respectively, 15 Rajiv Bahl et al16 in 2005 at New Delhi showed 26% positivity, Satarupa Mullick et al6 in 2014 at Kolkata showed 25.2%, Manohar Badur et al 14 in 2015 at Tirupati showed 25.67%. In a study conducted by Shobha Ram et al(1990) at Chandigarh observed M:F of 3.3:1, for rotavirus infection. 17 The present study correlates with study by P. Sarvanan et al(2004), 18 Pune, 19 Karachi20 and Turkey21 which detected rotavirus antigen in equal frequency in both males and females.
Seasonwise distribution of Rotavirus diarrhoea:
In the present study 88% cases were detected in the winter, 3.44% in summer and 10% during rainy season.
In a study conducted in southern Orissa from Jan 2007-Dec 2010 among children less than 5 years of age; higher prevalence of diarrhoea was noticed in July to October that is during monsoon period.22
M.R. Saha et al (1984) at Calcutta detected frequent rotavirus infection during cooler months of the year.23 A study at Nepal by M. Shariff et al(2003) observed that rotavirus infections occurred throughout the year.24
A study conducted at Vietnam reported that rotavirus infection occuredall round the year, with a peak in winter season.25
A study demonstrated that the degree of sensitivity and specificity of LA test showed a roughly linear relationship with the degree of ELISA sensitivity and specificity, and the positive predictive value of the test was high.26 According to Raj et al, the LA kit studied was a specific and rapid method. While it may be useful in certain situations, such as in outbreaks, its low sensitivity can make it unsuitable for use in routine clinical practice.27
Conclusion
Incidence of rotavirus cases in and around Hubballi is 29% in children less than 5 years of age. The study shows that maximum rotavirus cases were seen in the age group of 6-24 months. As the samples collected were from unvaccinated children, it also relates the importance of vaccination which could prevent dehydration resulting from rotavirus infection.Also, a rapid diagnosis of these infections with EIA, may be valuable in the formulation of prognosis for children with acute diarrhoea and may prevent the irrational use of antibiotic therapy and prolonged hospitalization.
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